Merged Beam MALDI TOF-TOF Using Ion-Ion Reactions to Determine Structure of Biological Molecules Summary The ultimate goals of this research are practical techniques for MALDI-TOF MS-MS on biologically significant molecules using electron transfer dissociation (ETD) of positive ions and proton transfer dissociation (PTD) of negative ions to determine molecular structure. This technology will provide the ability to study ion-ion collision processes under single collision conditions with direct measurement of the results in terms of charge transfer and fragmentation. Reaction cross sections for competing processes will be measured for several types of ion-ion collision processes. The emphasis in the fundamental studies is on reactions that lead to excited ions that dissociate following collision to produce structurally informative fragment ions. Singly charged precursors may be analyzed by employing double charge transfer to first produce excited neutrals that fragment efficiently, and then ionize the fragments by a second collision to produce fragment ions with charge opposite that of the precursor. In addition to fundamental knowledge, this research will provide practical techniques for determining molecular structure of singly and doubly charged molecular ions produced by MALDI. The approach should be applicable to a wide variety of molecules including intact proteins, peptides, oligonucleotides, oligosaccharides, glycoconjugates, lipids, and metabolites, and may provide speed, sensitivity, and specificity for identification and structural elucidation of unknowns that surpasses that available with electrospray ionization and ion traps. The instrument features addition of a second pulsed ion source to a recently developed high- performance MALDI-TOF-TOF MS-MS system. Pulsed beams from the two sources are merged within a collision cell with the pulses accurately synchronized in time, and with the relative velocity of the beams accurately determined and very near zero. The proposed apparatus will allow sensitive measurements of reactions between ions of opposite polarity and between ions and excited neutrals at low translational energies not previously accessible. The internal temperature (or excitation) of the reactants may be quite high but the translational temperatures are very low leading to very high cross sections for these processes. Collisions with reduced mass of 100 Da and relative velocity of 1 m/s correspond to translational energy of 5x10-7 eV, and translational temperature of 6x10-3 K. We are not aware of any previous work on ion-ion collision processes under these conditions. PUBLIC HEALTH RELEVANCE (provided by applicant): The "Holy Grail" of mass spectrometry for biological applications is an instrument that provides accurate molecular weight and complete, unambiguous sequence, including unusual amino acids and post- translational modifications, on proteins present at trace levels in complex mixtures. It should also provide similar information on mass and structure of other biologically significant molecules, including metabolites, lipids, oligosaccharides, glycoconjugates, oligonucleotides, and peptides. This may be it.